Diagnostic product and process for the detection of niacin production by mycobacteria

ABSTRACT

A diagnostic product for the detection of niacin produced by &#39;&#39;&#39;&#39;human&#39;&#39;&#39;&#39; Mycobacterium tuberculosis is prepared by impregnating a plurality of individual but separated zones on a paper strip with a series of reagents which include (1) an alkali metal salt of p-aminobenzoic acid; (2) sodium or potassium thiocyanate; (3) a crystalline acid such as citric, oxalic or malonic; and (4) chloramine-T. The desired diagnostic test employing the impregnated paper strip is performed by bringing the strip into contact and in a sealed test tube with an extract of the culture to be tested. If niacin is present the reaction of the several reagents with the niacin leads to color formation as a positive test for the presence of niacin.

United States Patent Inventors Donald P. Kronish Rockaway; William D. Young, Jr-, Montclalr, both of NJ. App]. No. 834,424 Filed June 18, 1969 Patented Oct. 26, 1971 Assignee Warner-Lambert Company Morris Plains, NJ.

DIAGNOSTIC PRODUCT AND PROCESS FOR THE DETECTION OF NIACIN PRODUCTION BY MYCOBACTERIA 11 Claims, 1 Drawing Fig.

US. Cl l95/103.5 R,

23/230 Int. Cl Cl2k 1/06 Field of Search 195/1035 AREA l l3.5 mm

AREA 2 AREA 3 l2.5 mm

AREA 4 AREA 5 AREA 6 AREA 7 l3.5 mm

AREA 8 l2.5 mm

AREA 9 References Cited OTHER REFERENCES Bailey et al., Diagnostic Microbiology 2nd Ed. Pages 191 and 192 1966) Primary Exam iner-Alvin E. Tanenholtz Attorneys-Albert H. Graddis, Henry E. Millson, Jr., Frank S.

Chow, Neil D. Edwards and Anne M. Kelly ABSTRACT: A diagnostic product for the detection of niacin produced by human" Mycobacterium tuberculosis is prepared by impregnating a plurality of individual but separated zones on a paper strip with a series of reagents which include (1) an alkali metal salt of p-aminobenzoic acid; (2) sodium or potassium thiocyanate; (3) a crystalline acid such as citric, oxalic or malonic; and (4) chlorarriine-T. The desired diagnostic test employing the impregnated paper strip is performed by bringing the strip into contact and in a sealed test tube with an extract of the culture to be tested. If niacin is present the reaction of the several reagents with the niacin leads to color formation as a positive test for the presence of niacin.

ZONE A ZONE 8 ZONE 83 mm ZONE ZONE

ZONE

PATENTEDUET 26 I9?! 3.616258 AREA l ZONE l3.5 mm A AREA 2 AREA 3 ZONE l2.5 mm B AREA 4 AREA 5 ZONE 83 mm 5 mm c AREA 6 AREA 7 ZONE l3.5 mm D AREA 8 ZONE l2.5 mm E AREA 9 ZONE 6.5 mm F m m INVENTOR.

Donald P. Kronlah y William D. Young, Jr.

Agent DIAGNOSTIC PRODUCT AND PROCESS FOR THE DETECTION OF NIACIN PRODUCTION BY MYCOBACTERIA BACKGROUND OF THE INVENTION The ability of human tubercle bacilli to produce niacin distinguishes Mycobacterium tuberculosis from nonhuman" mycobacteria.

Methods for detecting niacin produced by bacteria are known. The original Konno test (Science Vol. 124: 985, 1956) involves the transfer of bacilli to a test tube containing 4 percent alcoholic aniline; the addition of percent cyanogen bromide produces a yellow color if adequate concentrations of niacin are present. A modification of the Konno test was developed by Runyon (Am. Rev. Tuberc. and Pulm. Dis. 79: 663-665, 1959) also utilizes an extract of the culture to be tested. However, the use of the highly toxic color developing reagent cyanogen bromide is still necessary.

Other test procedures have been reported (Kilburn, J.O. and Kubica, G.P. Tech. Bull. Registry of Medical Technologists 38: 244-246, Sept., 1968) wherein efforts were made to supply the necessary reagents in the form of an impregnated strip. Stability problems with the materials described made their usefulness somewhat limited.

Thus, the need for a stable, sensitive diagnostic product,

which can be used with safety for the facile detection of niacin 7 production by human" mycobacteria, still exists.

SUMMARY OF THE INVENTION The present invention provides a stable, sensitive diagnostic product for the detection by color development of niacin produced by human mycobacteria. The novel diagnostic product is in the form of a strip of bibulous material impregnated with a series of reagents which react with niacin to produce a characteristic color. These diagnostic test strips are stable for 12 minutes at room temperature.

With the test strip of this invention, it is possible to detect the presence of as little as 3.1 micrograms of niacin in a 0.6 ml. solution of an extract of the culture being tested. Since the test is performed in a sealed test tube, danger from toxic reagents is avoided.

DESCRIPTION OF THE DRAWING The accompanying drawing is a diagrammatic representation of the bibulous test strip of the present invention. The strip is divided longitudinally into a series of areas impregnated with a particular reagent and separated from each other by unimpregnated areas. Thus, area 1 is impregnated with zone A Reagent, area 3 with Zone B Reagent, area 5 with Zone C Reagent and area 7 with Zone D Reagent; untreated areas 2, 4 and 6 separate the treated areas. In the preferred embodiment shown, area 8 is impregnated with Zone E Hydrophobic Barrier and area 9 with Zone F Dye.

DESCRIPTION OF THE INVENTION The diagnostic product of the present invention is prepared by impregnating a plurality of individual but separated zones of a suitable bibulous material, said zones being identified for convenience in relation to the specific reagent applied as Zones A, B, C and D with the following reagent media:

Zone A. p-Aminobenzoic acid.

This is supplied in the form of an aqueous or an aqueous alcoholic solution of an alkali metal salt of paminobenzoic acid having a measured pH of from about 7.5 to 12;

Zone B. Potassium thiocyanate.

This is applied as an aqueous solution of sodium or potassium thiocyanate;

Zone C. A crystalline acid in dry form.

This may be applied as an aqueous solution of a crystalline acid such as citric, oxalic or malonic acid; and

Zone D. Chloramine-T.

This can be applied as an aqueous solution of chloramine- The several reagent Zones A, B, C and D can be placed on the bibulous material in a combination of positions relative to each other as more fully described below.

In order to utilize the diagnostic test product and to test for niacin production by a particular organism, an aqueous or saline extract of a culture of the organism to be tested is placed in a 13X 100 mm. test tube or similar container. The reagent-impregnated diagnostic test strip is inserted into the extract fiuid and the container is immediately sealed. Capillary migration of reagents andgentle agitation, at room temperature, permits interaction of the reagents with any niacin present in the extract. The development of a specific color reaction in about 15 to 20 minutes is a positive indication of the presence of niacin. The color development reactions which take place when niacin is present are represented by:

-COOH COOH ClCEN NIACIN CYANOGEN CHLORIDE GLUTACONIC ALDEHYDE DERIVATIVE l COOH Aminobenzole acid pr ary aromatic amino Chrornogen The cyanogen chloride is formed by the reaction of the chloramine-T with the thiocyanate present on the test strip at an acid pH.

Since the container in which the test is carried out is sealed during the the test of the toxic reaction products, the hazards of prior art methods involving handling of such toxic reaction products are completely avoided. For disposal, the reactants are further treated after completion of the test to avoid toxicity problems.

In addition to ease of use, safety, and sensitivity, the diagnostic product of this invention is, as noted, stable for 12 months at room temperature.

The p-aminobenzoic acid reagent solution utilized for impregnating Zone A is prepared by dissolving from about 0.5 to 20 g. ofan alkali metal salt of p-aminobenzoic acid in about 20 to 100 ml., preferably about 30 to 50 ml. of distilled water, to which is then added a sufficient amount of a miscible organic solvent, such as ethanol, to bring the volume of the final solution to about 100 ml. The concentration of the alcohol in the final solution can be from about 10 to percent but is preferably from about 40 to 60 percent by volume. A sufficient amount of an alkaline agent which does not interfere with the diagnostic test is added to bring the pH of the final solution to a measured pH of from about 7.5 to 12, preferably about 9 to l2. Suitable alkali metal salts of p-aminobenzoic acid which can be used include sodium and potassium salts; of these the sodium salt is preferred. Suitable pH-modifying agents which do not interfere with the diagnostic test include such alkaline materials as sodium, potassium or ammonium hydroxide. Preferably about 0.lN sodium hydroxide solution is used.

In a preferred embodiment of this invention, the paminobenzoic acid salt employed is dissolved in approximately all of the distilled water to be used, the pH is adjusted to about 7.5 to 12, most preferably to about 10.5 to 10.7 and ethanol, the preferred alcohol solvent, is then added. The measured pH of this final solution falls between about-9 to l2.

The thiocyanate reagent solution applied to Zone B is prepared by dissolving from about 40 to 80 g., preferably about 50 to 70 g., of sodium or potassium thiocyanate in less than 100 ml. of distilled water and then bringing the volume of the solution to 100 ml. by adding further distilled water.

The acid reagent solution applied in Zone C is prepared by dissolving from about 30 to 70 g. preferably from about 40 to 60 g., of citric acid, oxalic acid or malonic acid, or a mixture of these acids, in distilled water and then adding sufficient additional distilled water to bring the volume of the final solution to 100 ml.

The chloramine-T reagent solution applied to form Zone D is prepared by dissolving from about 10 to 60 g. preferably from about 30 to 60 g., of chloramine-T in a lesser volume of distilled water and then adding further distilled water to bring the volume of the solution to 100 ml.

The above-described reagent solutions are applied to Zones A, B, C and D, respectively, of the bibulous material. If desired, they can be applied in several applications to the respective zone in order to apply and to provide the specific zones with a residue of the following amounts of reagent materials after the solvents in which they are applied have evaporated:

Broad Range in mg. Preferred Range in mg.

Zone A Reagent about 0.2 to 6 about i to Zone B Reagent about 0.6 to 20 about 6 to l3 Zone C Reagent about 0.2 to i0 about 2 to 3 Zone D Reagent about 1 to 20 about 2 to lo As previously stated, the above-described reagent zones may be applied to the bibulous test strip in a variety of combinations either side-by-side in alphabetical sequence or in random sequence. Color formulation, as an indication of a positive result, will vary depending on the order of placement chosen for the several reagents. The following chart lists the operable combinations possible, along with the corresponding color reactions indicating a positive or negative result:

N egatlve Sequence of color reaction reagent solutions Positive color reaction NOTE.-A=p-aminobenzoic acid salt solution; B=thiocyanate solution; C=acid solution; D=eh1oramine-T solution. I

Those combinations which yield a yellow solution as indicating a positive niacin reaction are preferred, since this color reaction is similar to that obtained using classical test procedures. Within this category, the ABCD and ACBD sequences are the more sensitive, with the ABCD sequence being the particularly preferred product overall. Of those sequence combinations where orange-pink" indicates a positive niacin reaction, the CDBA,.BACD, BDAC and CBDA are preferred, with the CDBA the most preferred in this category.

Optionally, one end of the impregnated test strip may be coated with a hydrophobic material to serve as a means for handling the test strip while avoiding any contamination of the reagent zones. FOr example, a zone impregnated with a hydrophobic barrier composition (hereinafter designated Zone E) may be placed at one end of the strip adjacent to the outermost reagent zone. For identification or handling a zone impregnated with a dye may also be provided (hereinafter designated Zone F), contiguous to the above-described hydrophobic barrier Zone E. This dyed zone can also be topcoated with a hydrophobic barrier composition such as that utilized for the coating of Zone E.

For the hydrophobic barrier, a composition is used which will prevent the leaching of the culture extract and reagent reactants upward to the end of the bibulous material or into the colored zone, if such a zone is present. The barrier composition should, of course, be chemically inert in this system. Substances which will form a waterproof barrier of this type include waxes, lacquers and plastic materials, particularly the colorless polymerized methyl methacrylate coating composition sold under the trade name KRYLON 150 CRYSTAL CLEAR, by Krylon, lnc., Norristown, Pa. The KRYLON material is particularly preferred. it is supplied as a solution in a toluene vehicle and may be diluted for ease of application with additional toluene or other diluents such as ethyl, methyl, or propyl alcohol USP.

it has been found that the desired hydrophobic effect can be obtained utilizing commercially available KRYLON solution diluted with up to about 25 percent of additional diluent.

Any suitable dye which will color the bibulous material sufficiently to distinguish the end of the diagnostic strip to be handled from the colorless reagent zones which are to be inserted into the culture extract may be used for the dye solution identified as Zone F. For example, we have found that dyes such as Sudan lV (Color Index No. 258 and Certification No. N218) or Sudan Red GGA in a suitable volatile solvent, i.e. xylene, may be used. Many other dyes are also suitable. The preferred dye solution which is applied contains from about 0.05 to about 0.8 g., preferably from about 0.1 to about 0.5 g. of the dye in about lOO ml. of volatile solvent.

The bibulous materials suitable for use in producing the product of this invention are those materials which by means of capillary action are able to hold liquid. Such materials include filter paper, felt, porous ceramic strips, woven or matted glass fiber and the like. A particularly preferred form of paper for this purpose is Eaton-Dikeman No. 623 filter paper (70 lbs).

in the preferred embodiment of our invention, the diagnostic product is prepared by impregnating separate zones of Eaton-Dikeman No. 623 filter paper with successive applica tions of the reagents listed below until there has been deposited on the particular zone the preferred amounts of each reagent after solvent evaporation. The preferred order in which the zones are applied is ABCDEF, as shown in the accompanying drawing, with each of zones ABCD being separated from each other by an intermediate untreated area 'and with Zones E and F immediately following and contiguous with Zone D without an untreated area in between.

The following examples are included further to illustrate this invention:

EXAM PLE l ABCDEF Sequence A. Preparation ofZones A Reagent Solution, p-Aminobenzoic Acid Salt.

l0 grams of p-aminobenzoic acid, sodium salt is dissolved in distilled water, and the solution is brought to a volume of 50 ml. with additional distilled water. The pH of the solution is adjusted to l0.5-l(}.7, by adding 0.1N NaOH solution as needed. The solution is brought to 100 ml. with percent ethanol. B. Preparation of the Zone B Reagent Solution, Potassium -Thiocyanate.

60 grams of potassium thiocyanate is dissolved in distilled water and the solution is brought to a vdiiin'ibr l(l(l with additional distilled water. C. Preparation of the Zone C Reagent Solution, Citric Acid.

50 grams of citric acid is dissolved in distilled water and the solution is brought to 100 ml. with additional distilled water. This material becomes cold as it dissolved and it must be warmed to room temperature.

D. Preparation of the Zone D Reagent Solution, Chloramine- T.

50 grams of chloramine-T is dissolved in distilled water, with heating to 58-60 C. to insure complete dissolution. Sufficient distilled water is added to bring the volume to 100 ml, while maintaining the temperature at 58-60 C. to prevent recrystallization.

E. Preparation ofZone E Hydrophobic Barrier Composition.

85 ml. of KRYLON 150 CRYSTAL CLEAR is diluted with ml. of ethyl alcohol, USP.

F. Preparation of Zone F Dye Solution.

0.3 grams of Sudan Red GGA is dissolved in xylene and the solution is brought to a volume of 100 ml. with additional xylene.

EXAMPLE 2 Application of Diagnostic Reagents to Bibulous Material In producing the strips, a continuous sheet of Eaton- Dikeman Filter Paper No. 623(70 lbs.), 83 mm. in width, was employed. The paper was divided into nine separate areas having the width shown in the accompanying drawing.

Areas 2, 4 and 6 are untreated. Zone A reagent solution is applied to Area 1, twice one each side of the paper, allowing drying time between the first and second application to each side. This provides about 3.4 mg. of sodium p-aminobenzoic acid in each test strip. Zone B reagent solution is applied to Area 3, twice on each side of the paper, allowing drying time between the first and second application to each side. This application provides about 9 mg. of potassium thiocyanate on each test strip. Care must be taken not to allow Zone A and Zone B reagent solutions to mix. Zone C reagent solution is applied to Area 5, once on each side of the paper which deposits about 2.8 mg. of citric acid on the test strip. Zone D reagent solution, maintained at 58-60 C. during application, is applied to Area 7, twice on each side of the paper, allowing drying time between the first and second application to each side which deposits about 8 mg. of chloramine-T on each test strip. Extreme caution must be taken to avoid any mixing or contact between Zone B reagent solution and Zone D reagent solution: after each application to the zones, the paper should be dried in a fume hood, since if these reagents are mixed, a highly toxic gas, cyanogen chloride is formed. Zone F reagent solution is applied to Area 9, once on one side of the paper and allowed to dry. Zone E reagent solution is applied to Areas 8 and 9, once on each side of the paper, coating both areas, and allowed to dry. After all areas of the paper have dried thoroughly, cut into Arinch (6.3 mm.) strips, each containing the nine areas impregnated with solutions herein described.

EXAMPLE 3 Use of the Diagnostic Composition Method of Use Place 0.6 ml. of a saline extract of the culture to be tested (Mycobacterium species) in a 13 X100 mm. or similar size tube. Add one diagnostic test strip prepared according to example 2, with Zone A (the end opposite the dyed marker zone) dipped into saline solution, the stopper immediately with a paraffin coated cork stopper. Allow to stand for 15 to minutes at room temperature with occasional gentle shaking. A positive test for niacin is indicated by the development of an orange to yellow color in the solution. A negative test is essentially colorless. Any color on the strip itself is disregarded. For disposal, one-half to 1 ml. of 10 percent aqueous NaOH should be added to each tube, restoppered, mixed and allowed to stand one-halfto 1 hour before autoclaving.

EXAMPLE 4 The procedures of examples 1 and 2 are followed except that the reagent zones are applied to the paper in the sequence A, C, B, D; the same order and procedure is followed for Zones E and F as in examples 1 and 1 EXAMPLE 5 The procedures of examples 1 and 2 are followed except that the reagent zones are applied to the paper in the sequence C, D, B, A; the same order and procedure is followed for Zones E and F as in examples 1 and 2.

EXAMPLE 6 The procedures of examples 1 and 2 are followed except that the reagent zones are applied to the paper in the sequence B, A, C, D; the same order and procedure is followed for Zones E and F as in examples 1 and 2.

EXAMPLE 7 The procedures of examples 1 and 2 are followed except that the reagent zones are applied to the paper in the sequence B, D, A, C; the same order and procedure is followed for Zones E and F as in examples 1 and 2.

EXAMPLE 8 The procedures of examples 1 and 2 are followed except that the reagent zones are applied to the paper in the sequence C, B, D, A; the same order is followed for Zones E and F, as in examples 1 and 2.

EXAMPLE 9 The procedure of example 3 is followed, using the test strip as prepared in example 5. As a positive indication of the presence of niacin, the strip becomes orange to pink and a yellow cross-reaction also appears on the test strip. The positive or negative color reactions of the test strips of examples 6, 7 and 8 can be found above.

We claim:

1. A diagnostic composition for the positive detection of niacin production by mycobacteria, which comprises a bibulous material containing at least four reagent-impregnated zones, separated one from the other by an untreated area of the bibulous material, wherein:

A. Zone A contains from about 0.2 to about 6 mg. of an alkali metal salt of p-aminobenzoic acid;

B. Zone B contains from about 0.6 to about 20 mg. of sodiurn or potassium thiocyanate;

C. Zone C contains from about 0.2 to about 10 mg. of a crystalline acid selected from the group citric acid, oxalic acid and malonic acid; and

D. Zone D contains from about 1 to about 20 mg. of

chloramine-T;

said zones being arranged in an order adjacent to each other which will give a positive reaction on contact with an aqueous or saline extract ofa niacin-producing culture.

2. A diagnostic composition according to claim 1, wherein an additional Zone E, impregnated with a chemically inert hydrophobic barrier composition, is positioned contiguous to the untreated area following the outermost reagent impregnated zone.

3. A diagnostic composition according to claim 2, wherein an additional Zone F, impregnated with a dye solution and topcoated with a hydrophobic barrier composition is contiguous to Zone E.

4. A diagnostic composition for the positive detection of niacin production by mycobacteria, which comprises a bibulous material containing at least four reagent-impregnated zones, separated one from the other by an untreated area of the bibulous material, wherein:

A. Zone A contains from about 1.0 to about 4 mg. of an alkaii metal salt of p-aminobenzoic acid;

B. Zone B contains from about 6.0 to about 13 mg. of sodium or potassium thiocyanate;

C. Zone C contains from about 2.0 to about 3.0 mg. of a crystalline acid selected from the group citric acid, oxalic acid, or malonic acid; and

D. Zone D contains from about 2 to about mg. of

chloramine-T;

said zones being arranged in an order adjacent to each other which will give a positive reaction on contact with an aqueous or salineextract of a niacin-producing culture. A

5. A diagnostic composition according to claim 4, wherein an additional Zone E, positioned contiguous to the untreated area following the outermost reagent-impregnated zone, is impregnated with a chemically inert hydrophobic barrier composition comprising:

A. from about 75 to about 100 ml. of an acrylic coating composition; and

B. from about 0 to about 25 ml. of a hydrocarbon thinner for said coating composition.

6. A diagnostic composition according to claim 5, wherein an additional Zone F, contiguous to Zone E, is impregnated with a dye solution comprising an about lOO-ml. solution of from about 0.05 to about 0.8 g. of a dye; and wherein said dyeimpregnated zone is topcoated with a hydrophobic barrier composition.

7. A diagnostic composition for the positive detection of niacin production by mycobacteria, which comprises a bibulous material containing at least four reagent-impregnated zones, separated one from the other by an untreated area of the bibulous material, wherein:

A. Zone A contains about 3.4 mg. of the sodium saltof paminobenzoic acid;

B. Zone B contains about 9 mg. of potassium thiocyanate;

C. Zone C contains about 2.8 mg. ofcitric acid; and

D. Zone D contains about 8 mg. of chloramine-T', wherein the reagent-impregnated zones are positioned on the bibulous material in the sequence Zone A, Zone B, Zone C and Zone D, and wherein Zone A is contiguous only to the untreated area preceding Zone E; Zone B is contiguous only to the untreated area following Zone A and the untreated area preceding Zone C; Zone C is contiguous only to the untreated area foliowing Zone B and the untreated area preceding Zone D; Zone D is contiguous to the untreated area following Zone C.

8. A diagnostic composition according to claim 7, wherein an additional Zone E, positioned contiguous to Zone D, is im-v pregnated with a chemically inert hydrophobic barrier composition comprising about mg. of a methyl methacrylate coating composition and about 15 ml. of ethyl alcohol.

9. A diagnostic composition according to claim 8, wherein an additional Zone F, contiguous to Zone E, is impregnated with a dye solution comprising an about l00-ml. solution of about 0.1 g. of a dye; and wherein said dye-impregnated zone is topcoated with a hydrophobic barrier composition comprising about 85 ml. of a methyl methacrylate coating composition and about 15 ml. of ethyl alcohol.

10. A diagnostic composition according to claim 7, wherein the reagent-impregnated zones are positioned on the bibulous material in the sequence Zone C, Zone D, Zone 8 and Zone A, and wherein Zone C is contiguous only to the untreated area preceding Zone D; Zone D is a contiguous only to the untreated area following Zone C and the untreated area preceding Zone B; Zone B is contiguous only to the untreated area following Zone D and the untreated area preceding Zone A; Zone A is contiguous to the untreated area following Zone B.

11. A process for the detection of niacin production by mycobacteria comprising:

A. Inserting the diagnostic preparation of claim 1 into a test tube containing an aqueous or saline extract of the culture to be tested;

B. Scaling the mouth of the test tube with an inert stopper material;

C. Maintaining the sealed test tube at room temperature with gentle agitation;

D. Allowing at least 15 to 20 minutes to elapse for color to develop as a positive indication of the presence of niacin. 

2. A diagnostic composition according to claim 1, wherein an additional Zone E, impregnated with a chemically inert hydrophobic barrier composition, is positioned contiguous to the untreated area following the outermost reagent impregnated zone.
 3. A diagnostic composition according to claim 2, wherein an additional Zone F, impregnated with a dye solution and topcoated with a hydrophobic barrier composition is contiguous to Zone E.
 4. A diagnostic composition for the positive detection of niacin production by mycobacteria, which comprises a bibulous material containing at least four reagent-impregnated zones, separated one from the other by an untreated area of the bibulous material, wherein: A. Zone A contains from about 1.0 to about 4 mg. of an alkali metal salt of p-aminobenzoic acid; B. Zone B contains from about 6.0 to about 13 mg. of sodium or potassium thiocyanate; C. Zone C contains from about 2.0 to about 3.0 mg. of a crystalline acid selected from the group citric acid, oxalic acid, or malonic acid; and D. Zone D contains from about 2 to about 10 mg. of chloramine-T; said zones being arranged in an order adjacent to each other which will give a positive reaction on contact with an aqueous or saline extract of a niacin-producing culture.
 5. A diagnostic composition according to claim 4, wherein an additional Zone E, positioned contiguous to the untreated area following the outermost reagent-impregnated zone, is impregnated with a chemically inert hydrophobic barrier composition comprising: A. from about 75 to about 100 ml. of an acrylic coating composition; and B. from about 0 to about 25 ml. of a hydrocarbon thinner For said coating composition.
 6. A diagnostic composition according to claim 5, wherein an additional Zone F, contiguous to Zone E, is impregnated with a dye solution comprising an about 100-ml. solution of from about 0.05 to about 0.8 g. of a dye; and wherein said dye-impregnated zone is topcoated with a hydrophobic barrier composition.
 7. A diagnostic composition for the positive detection of niacin production by mycobacteria, which comprises a bibulous material containing at least four reagent-impregnated zones, separated one from the other by an untreated area of the bibulous material, wherein: A. Zone A contains about 3.4 mg. of the sodium salt of p-aminobenzoic acid; B. Zone B contains about 9 mg. of potassium thiocyanate; C. Zone C contains about 2.8 mg. of citric acid; and D. Zone D contains about 8 mg. of chloramine-T; wherein the reagent-impregnated zones are positioned on the bibulous material in the sequence Zone A, Zone B, Zone C and Zone D, and wherein Zone A is contiguous only to the untreated area preceding Zone B; Zone B is contiguous only to the untreated area following Zone A and the untreated area preceding Zone C; Zone C is contiguous only to the untreated area following Zone B and the untreated area preceding Zone D; Zone D is contiguous to the untreated area following Zone C.
 8. A diagnostic composition according to claim 7, wherein an additional Zone E, positioned contiguous to Zone D, is impregnated with a chemically inert hydrophobic barrier composition comprising about 85 mg. of a methyl methacrylate coating composition and about 15 ml. of ethyl alcohol.
 9. A diagnostic composition according to claim 8, wherein an additional Zone F, contiguous to Zone E, is impregnated with a dye solution comprising an about 100-ml. solution of about 0.1 g. of a dye; and wherein said dye-impregnated zone is topcoated with a hydrophobic barrier composition comprising about 85 ml. of a methyl methacrylate coating composition and about 15 ml. of ethyl alcohol.
 10. A diagnostic composition according to claim 7, wherein the reagent-impregnated zones are positioned on the bibulous material in the sequence Zone C, Zone D, ZOne B and Zone A, and wherein Zone C is contiguous only to the untreated area preceding Zone D; Zone D is a contiguous only to the untreated area following Zone C and the untreated area preceding Zone B; Zone B is contiguous only to the untreated area following Zone D and the untreated area preceding Zone A; Zone A is contiguous to the untreated area following Zone B.
 11. A process for the detection of niacin production by mycobacteria comprising: A. Inserting the diagnostic preparation of claim 1 into a test tube containing an aqueous or saline extract of the culture to be tested; B. Sealing the mouth of the test tube with an inert stopper material; C. Maintaining the sealed test tube at room temperature with gentle agitation; D. Allowing at least 15 to 20 minutes to elapse for color to develop as a positive indication of the presence of niacin. 